Mitochondrial diseases are devastating disorders for which there is no cure and no proven treatment. About 1 in 2000 individuals are at risk of developing a mitochondrial disease sometime in their lifetime. Half of those affected are children who show symptoms before age 5 and approximately 80% of these will die before age 20. The mortality rate is roughly that of cancer. The human suffering imposed by mitochondrial and metabolic diseases is enormous, yet much work is needed to understand the genetic and environmental causes of these diseases. Mitochondrial genetic diseases are characterized by alterations in the mitochondrial genome, as point mutations, deletions, rearrangements, or depletion of the mitochondrial DNA (mtDNA). The mutation rate of the mitochondrial genome is 10-20 times greater than of nuclear DNA, and mtDNA is more prone to oxidative damage than is nuclear DNA. Mutations in human mtDNA cause premature aging, severe neuromuscular pathologies and maternally inherited metabolic diseases, and influence apoptosis.[unreadable] The primary goal of this project is to understand the contribution of the replication apparatus in the production and prevention of mutations in mtDNA. Since the genetic stability of mitochondrial DNA depends on the accuracy of DNA polymerase gamma (pol gamma), we have focused this project on understanding the role of the human pol gamma in mtDNA mutagenesis. Human mitochondrial DNA is replicated by the two-subunit gamma, composed of a 140 kDa subunit containing catalytic activity and a 55 kDa accessory subunit. The catalytic subunit contains DNA polymerase activity, 3'-5' exonuclease proofreading activity, and 5'dRP lyase activity required for base excision repair. As the only DNA polymerase in animal cell mitochondria, pol gamma participates in DNA replication and DNA repair. The 140 kDa catalytic subunit for pol gamma is encoded by the nuclear POLG gene. To date there are nearly 90 pathogenic mutations in POLG that cause a wide spectrum of disease including Progressive external ophthalmoplegia (PEO), parkinsonism, premature menopause, Alpers syndrome, mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) or sensory ataxic neuropathy, dysarthria, and ophthalmoparesis (SANDO).[unreadable] PEO is a mitochondrial disorder associated with mtDNA deletions and point mutations. PEO is characterized by late onset (between 18 and 40 years of age) bilateral ptosis and progressive weakening of the external eye muscle, resulting in blepharoptosis and ophthalmoparesis, proximal muscle weakness and wasting as well as exercise intolerance. The disease is often accompanied by cataract, hypogonadism, dysphagia, hearing loss and may, within several years, lead to development of neuromuscular problems. Neurological problems may include depression or avoidant personality. Skeletal muscles of PEO patients present red ragged fibers and lowered activity of respiratory chain enzymes. PEO can be inherited in an autosomal dominant or recessive manner.[unreadable] Alpers syndrome is an autosomal recessive mitochondrial DNA depletion disorder that affects children and young adults. It is a devastating disease characterized by psychomotor retardation, hepatic failure and intractable seizures, as well as tissue-specific mtDNA depletion. Alpers syndrome is 100% fatal with no cure available. This syndrome has been exclusively associated with mutations in POLG. Carriers for this disease are 1:250 with presentation occurring 1:100,000 to 250,000. We and others have identified over 30 pathogenic POLG mutations that cause Alpers in over 45 probands.[unreadable] Ataxia/neuropathy resulting from mutations in POLG is an autosomal recessive disorder affecting patients in their mid-teens to later years usually resulting in premature death. The disease is accompanied mainly by mtDNA deletions. The ataxia usually occurs in combination with various central nervous system features including myoclonus, epilepsy, cognitive decline, nystagmus, dysarthria, thalamic and cerebellar white matter lesions on MRI, and evidence of neuronal loss in discrete gray nuclei.[unreadable] Presently, there are nearly 90 pathogenic disease mutations in the POLG gene that cause PEO, ataxia-neuropathy and Alpers syndrome. To rapidly characterize the effects of these mutations, we have developed a versatile system that enables the consequences of homologous mutations, introduced in situ into the yeast mtDNA polymerase gene MIP1, to be evaluated in vivo in haploid and diploid cells. Overall, distinct phenotypes for expression of each of the mip1-PEO mutations were observed, including respiration-defective cells with decreased viability, dominant-negative mutant polymerases, elevated levels of mitochondrial and nuclear DNA damage and chromosomal mutations. Mutations in the polymerase domain caused the most severe phenotype accompanied by loss of mtDNA and cell viability, whereas the mutation in the exonuclease domain showed mild dominance with loss of mtDNA. Interestingly, the linker region mutation caused elevated mitochondrial and nuclear DNA damage. The cellular processes contributing to these observations in the mutant yeast cells are potentially relevant to understanding the pathologies observed in human mitochondrial disease patients.[unreadable] We have also identified a new genetic locus in progressive external ophthalmoplegia and biochemically characterized the defect.. We describe a heterozygous dominant mutation (c.1352GrA/p.G451E) in POLG2, the gene encoding the p55 accessory subunit of pol gamma, that causes progressive external ophthalmoplegia with multiple mtDNA deletions and cytochrome c oxidase (COX)?deficient muscle fibers. Biochemical characterization of purified, recombinant G451E-substituted p55 protein in vitro revealed incomplete stimulation of the catalytic subunit due to compromised subunit interaction. Although G451E p55 retains a wild-type ability to bind DNA, it fails to enhance the DNA-binding strength of the p140-p55 complex. In vivo, the disease most likely arises through haplotype insufficiency or heterodimerization of the mutated and wild-type proteins, which promote mtDNA deletions by stalling the DNA replication fork. The progressive accumulation of mtDNA deletions causes COX deficiency in muscle fibers and results in the clinical phenotype. [unreadable] Mitochondrial mutational spectra in human cells, tissues and derived tumors for bp 10,030?10,130 are essentially identical, suggesting a predominant mutagenic role for endogenous processes. We hypothesized that errors mediated by mitochondrial DNA polymerase gamma were the primary sources of mutations. Point mutations created in this sequence by human DNA pol gamma in vitro were thus compared to the eighteen mutational hotspots, all single base substitutions, previously found in human tissues. The set of concordant hotspots accounted for 83% of these in vivo mutational events. About half of these mutations are insensitive to prolonged heating of DNA during PCR and half increase proportionally with heating time at 98&#9702;C. Primary misincorporation errors and miscopying errors past thermal denaturing products such as deaminated cytosines (uracils) thus appear to be of approximately equal importance. For the sequence studied, these data support the conclusion that, endogenous error mediated by DNA pol gamma constitutes the primary source of mitochondrial point mutations in human tissues .